Title: Watching cooperative interactions between base and nucleotide excision repair proteins. PI: Bennett Van Houten, PhD Abstract/Summary This highly innovative project seeks to answer several fundamental questions regarding how DNA repair proteins work to protect the human genome from environmentally-induced DNA damage. This project uses an integrated approach combining biochemistry, single molecule analysis and highly innovative chemoptogenetic cell biology tools to study with high temporal and spatial resolution molecular hand-offs during DNA repair. We posit that key nucleotide excision repair proteins including UV-DDB, XPA, and XPC-RAD23B work in a dynamic way with specific base excision repair proteins to process oxidized bases in the context of chromatin. Specially, we will follow purified DNA repair proteins and/or proteins labeled from nuclear extracts as they interact at sites of damage on naked DNA and chromatinized DNA using a DNA tightrope optical platform. Building on preliminary data and premise that UV-DDB can change the register of specific lesions in the context of the nucleosome, we will test the paradigm shifting hypothesis that UV-DDB working in concert with other NER proteins is a general damage sensor and can stimulate APE1 and 11 mammalian DNA glycosylases activities on their respective oxidized DNA substrates. This project will develop and validate new genomic tools to place 8-oxoG adducts at defined sites throughout the genome to assess the how chromatin structure and chromatin remodelers effects repair. We will also develop and use several high-resolution fluorescent approaches including single particle tracking protocols (based on Halo- and SNAP-tags) to watch individual repair proteins arrive and process damage sites in real-time in living cells. Finally, we posit that UV-DDB and XPC-RAD23B work with thymine DNA glycosylase to alter methylation patterns in cells and ultimately change gene expression profiles. Together these approaches will give an unprecedented view of the complex process of DNA damage processing during repair and answer several key questions regarding damage recognition that have been intractable in the absence of super-resolution approaches. Completion of this project will have a long and lasting impact on the field.